EP2271665A1 - Stark verbrückte peptide aus der actinomadura namibiensis - Google Patents

Stark verbrückte peptide aus der actinomadura namibiensis

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Publication number
EP2271665A1
EP2271665A1 EP09728150A EP09728150A EP2271665A1 EP 2271665 A1 EP2271665 A1 EP 2271665A1 EP 09728150 A EP09728150 A EP 09728150A EP 09728150 A EP09728150 A EP 09728150A EP 2271665 A1 EP2271665 A1 EP 2271665A1
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EP
European Patent Office
Prior art keywords
formula
compound
alkyl
alkylene
labyrinthopeptin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP09728150A
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English (en)
French (fr)
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EP2271665B1 (de
Inventor
Mark Broenstrup
Hans Guehring
Holger Hoffmann
Joachim Wink
Roderich Suessmuth
Timo Schmiederer
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Sanofi SA
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Sanofi Aventis France
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Publication of EP2271665A1 publication Critical patent/EP2271665A1/de
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)

Definitions

  • conopeptides are useful for the treatment of pain, diabetes, multiple sclerosis and cardiovascular diseases and currently undergo preclinical or clinical development.
  • conopeptides are ⁇ -GI (sequence: ECCNPACGRHYSC * , * amidated, connectivity: 1-3,2-4) and ⁇ -GID (sequence: IR ⁇ CCSNPACRVNNOHVC, connectivity: 1-3,2-4), wherein O/Hyp is hydroxyproline and the connectivity indicates the position of the cysteine involved in each specific disulphide bonds, for example, first to third and second to fourth as in ⁇ -GID: S-S
  • Labyrinthopeptins A novel group of highly bridged peptides named Labyrinthopeptins has been discovered recently (European patent application EP06020980.6). The so-called Labyrinthopeptins exhibit a unique bridging motif across their peptide chain, as illustrated by the compound in formula below:
  • An embodiment of the present invention is a compound of the formula (I)
  • ⁇ A ⁇ is a group selected from
  • ⁇ B ⁇ is a group selected from
  • ⁇ C ⁇ is a group selected from
  • Ri is a group Ri' or a group wherein R 1 1 is H, C(O)-(C r C 6 )alkyl or C(O)-O-(C r C 6 )alkyl;
  • R 2 is OH, NH 2 , NH-(C 1 -C 6 )BIkYl, NH-(C r C 4 )alkylene-phenyl or NH-(Ci-C 4 )alkylene-pyridyl;
  • R 3 and R 4 are independently of each other H, (CrC 6 )alkyl, (d-C 6 )alkylene-C(O)NH 2 , (Ci-CeJalkylene-CCOJNHtCi-C ⁇ alkyl or tCi-CeJalkylene-CtOJNKd-C ⁇ alkylk, or R 3 and R 4 together with the S atoms to which they are attached form a disulfide group S-S;
  • n and n are independently of one another 0, 1 or 2;
  • R 3 and R 4 may not form a disulfide group S-S together with the S atoms to which they are attached;
  • Ri is preferably a group R 1 '.
  • Ri ' is preferably H.
  • R 2 is preferably OH.
  • R 3 and R 4 are preferably independently of each other H, (Ci-C 6 )alkyl, (Ci-C 6 )alkylene- C(O)NH 2 , or form a disulfide group S-S together with the S atoms to which they are attached. More preferred, R 3 and R 4 are H or form a disulfide group S-S together with the S atoms to which they are attached. Most preferred, R 3 and R 4 form a disulfide group S-S together with the S atoms to which they are attached.
  • compound (I) is characterized by a compound of the formula (II)
  • R 1 is R 1 1 or a group
  • R 1 ' is H, C(O)-(C r C 6 )alkyl or C(O)-O-(Ci-C 6 )alkyl, preferably H.
  • compound (I) is characterized by a compound of the formula (III)
  • Ri is R-i 1 or a group
  • R 1 ' is H, C(O)-(Ci-C 6 )alkyl or C(O)-O-(C r C 6 )alkyl, preferably H;
  • R 2 is OH, NH 2 , NH-(Ci-C 6 )-alkyl, N[(Ci-C 6 )-alkyll 2 , NH-(d-C 4 )-alkylene-phenyl or NH-(CrC 4 )-alkylene-pyridyl, preferably R 2 is H; and
  • R 3 and R 4 are independently from each other H, (CrC 6 )alkyl or (Ci-C 4 )-alkylene-C(O)NH 2 .
  • Labyrinthopeptins A3 are subsequently named Labyrinthopeptins A3.
  • compound (I) is characterized by a compound of the formula (IV)
  • R 1 is H, C(O)-(Ci-C 6 )alkyl or C(O)-O-(Ci-C 6 )alkyl, and
  • R 2 is OH, NH 2 , NH-(d-C 6 )-alkyl, N[(Ci-C 6 )-alkyl] 2 , NH-(C r C 4 )-alkylene-phenyl or NH-(Ci-C 4 )-alkylene-pyridyl, and
  • R 3 and R 4 are independently from each other H, (CrC 6 )alkyl or (Ci-C 4 )-alkylene-C(O)NH 2 .
  • Labyrinthopeptins A2 are named Labyrinthopeptins A2.
  • m and n are both 0, or m and n are both 2, or m is 0 and n is 2, or m is 2 and n is 0. Most preferred, m and n are both 0.
  • the present invention furthermore relates to all obvious chemical equivalents of the compounds of the formula (I) according to the invention.
  • These equivalents are compounds which exhibit only a slight chemical difference, and have the same pharmacological effect, or which are converted into the compounds according to the invention under mild conditions.
  • Said equivalents also include, for example, salts, reduction products, oxidation products, partial hydrolytic processes esters, ethers, acetals or amides of the compounds of the formula (I) as well as equivalents which the skilled person can prepare using standard methods and, in addition to this, all the optical antipodes and diastereomers and all the stereoisomeric forms.
  • the chiral centers in the compounds of the formula (I) can be present in the R configuration or in the S configuration.
  • the invention relates both to the optically pure compounds and to stereoisomeric mixtures, such as enantiomeric mixtures and diastereomeric mixtures.
  • Physiologically tolerated salts of compounds of the formula (I) are understood as being both their organic salts and their inorganic salts, as are described in Remington's Pharmaceutical Sciences (17th edition, page 1418 (1985)). Because of their physical and chemical stability and their solubility, sodium, potassium, calcium and ammonium salts are preferred, inter alia, for acid groups; salts of hydrochloric acid, sulfuric acid or phosphoric acid, or of carboxylic acids or sulfonic acids, such as acetic acid, citric acid, benzoic acid, maleic acid, fumaric acid, tartaric acid and p-toluenesulfonic acid, are preferred, inter alia, for basic groups.
  • the compounds of the formulae (I) to (IV) are characterized by the stereochemistry as shown for a compound of the formula (V), that is a compound of the formula (I), wherein
  • R 1 ' is H
  • R 2 are H
  • R 3 and R 4 together with the S atoms to which they are attached form a disulfide group
  • R 5 is H; R 6 is OH; and m and n are 0:
  • a further embodiment of the present invention is a compound of the formula (I), characterized by the formula (VII)
  • the invention also relates to a process for preparing a compound of the formula (I) according to claim 1 comprising a) fermenting the strain Actinomadura namibiensis (DSM 6313), or one of its variants and/or mutants, under suitable conditions in a culture medium until one or more of the compounds of the formula (I) accrue(s) in the culture medium, b) isolating a compound of the formula (I) from the culture medium, and c) derivatizing, where appropriate, the compound isolated in step b) and/or, where appropriate, converting the compound isolated in step b) or the derivative of compound isolated in step b) into a physiologically tolerated salt.
  • the compound isolated in step b) is characterized by formula (II) wherein m and n are both 0, Ri is Ri' or a group
  • R 1 ' is H, and R 2 is OH.
  • the compound isolated in step b) is Labyrinthopeptin A2 which subsequently derivatized in step c) to a compound of the formula (IV) wherein m and n are both 0,
  • R 1 is H
  • R 2 is OH, and R 3 and R 4 are independently of each other H, (CrC ⁇ alkyl, (CrC 6 )alkylene-C(O)NH 2 ,
  • the culture medium is a nutrient solution or a solid medium containing at least one customary carbon source and at least one nitrogen source as well as one or more customary inorganic salts.
  • the process according to the invention can be used for fermenting on a laboratory scale (milliliter to liter scale) and for fermenting on an industrial scale (cubic meter scale).
  • Suitable carbon sources for the fermentation are assimilable carbohydrates and sugar alcohols, such as glucose, lactose, sucrose or D-mannitol, as well as carbohydrate- containing natural products, such as malt extract or yeast extract.
  • nitrogen-containing nutrients are amino acids; peptides and proteins and also their breakdown products, for example casein, peptones or tryptones; meat extracts; yeast extracts; gluten; ground seeds, for example from corn, wheat, beans, soya or the cotton plant; distillation residues from producing alcohol; meat meals; yeast extracts; ammonium salts; nitrates.
  • inorganic salts are chlorides, carbonates, sulfates or phosphates of the alkali metals, the alkaline earth metals, iron, zinc, cobalt and manganese.
  • trace elements are cobalt and manganese.
  • Labyrinthopeptins Conditions which are especially suitable for forming the Labyrinthopeptins according to the invention are as follows: from 0.05 to 5%, preferably from 0.1 to 2.5%, yeast extract; from 0.2 to 5.0%, preferably from 0.1 to 2%, casitone; from 0.02 to 1.0%, preferably from 0.05 to 0.5%, CaCl2 x 2 H2O; from 0.02 to 1.5%, preferably from 0.05 to 0.7%, MgSO4 x 7 H2O and from 0.00001% to 0.001% cyanocobalamin.
  • the percentage values which are given are in each case based on the weight of the total nutrient solution.
  • the microorganism is cultured aerobically, that is, for example, submerged while being shaken or stirred in shaking flasks or fermenters, or on solid medium, where appropriate while air or oxygen is being passed in.
  • the microorganism can be cultured in a temperature range of from about 18 to 35°C, preferably at from about 20 to 32°C, in particular at from 27 to 30 0 C.
  • the pH range should be between 4 and 10, preferably between 6.5 and 7.5.
  • the microorganism is generally cultured under these conditions for a period of from 2 to 10 days, preferably of from 72 to 168 hours.
  • the micro- organism is advantageously cultured in several steps, i.e.
  • one or more preliminary cultures are initially prepared in a liquid nutrient medium, with these preliminary cultures then being inoculated into the actual production medium, i.e. the main culture, for example in a ratio by volume of from 1:10 to 1 :100.
  • the preliminary culture is obtained, for example, by inoculating the strain, in the form of vegetative cells or spores, into a nutrient solution and allowing it to grow for from about 20 to 120 hours, preferably for from 48 to 96 hours.
  • Vegetative cells and/or spores can be obtained, for example, by allowing the strain to grow for from about 1 to 15 days, preferably for from 4 to 10 days, on a solid or liquid nutrient substrate, for example yeast agar.
  • the Labyrinthopeptin derivatives can be isolated and purified from the culture medium using known methods and taking account of the chemical, physical and biological properties of the natural substances. HPLC was used to test the concentrations of the respective Labyrinthopeptin derivatives in the culture medium or in the individual isolation steps, with the quantity of the substance formed expediently being compared with a calibration solution.
  • the culture broth or the culture together with the solid medium is optionally lyophilized, and the Labyrinthopeptin derivatives are extracted from the lyophilizate using an organic solvent or a mixture of water and an organic solvent, preferably containing 50-90% organic solvent.
  • organic solvents are methanol and 2-propanol.
  • the organic solvent phase contains the natural substances according to the invention; it is concentrated, where appropriate, in vacuo and subjected to further purification.
  • the further purification of one or more compounds according to the invention is effected by chromatography on suitable materials, preferably, for example, on molecular sieves, on silica gel, on aluminum oxide, on ion exchangers or on adsorber resins or on reversed phases (RPs).
  • This chromatography is used to separate the Labyrinthopeptin derivatives.
  • the Labyrinthopeptin derivatives are chromatographed using buffered, basic or acidified aqueous solutions or mixtures of aqueous and organic solutions.
  • aqueous or organic solutions are understood as being all water-miscible organic solvents, preferably methanol, 2-propanol or acetonitrile, at a concentration of from 5 to 99% organic solvent, preferably from 5 to 50% organic solvent, or else all buffered aqueous solutions which are miscible with organic solvents.
  • the buffers which are to be used are the same as specified above.
  • Labyrinthopeptin derivatives are separated, on the basis of their differing polarities, by means of reversed phase chromatography, for example on MCI (adsorber resin, Mitsubishi, Japan) or Amberlite XAD (TOSOHAAS), or on other hydrophobic materials, for example on RP-8 or RP-18 phases.
  • the separation can be effected by means of normal-phase chromatography, for example on silica gel, aluminum oxide and the like.
  • Buffered, basic or acidified aqueous solutions are understood as being, for example, water, phosphate buffer, ammonium acetate and citrate buffer at a concentration of up to 0.5 M, as well as formic acid, acetic acid, trifluoroacetic acid, ammonia and triethylamine, or all commercially available acids and bases known to the skilled person, preferably at a concentration of up to 1%.
  • buffered aqueous solutions particular preference is given to 0.1 % ammonium acetate.
  • the chromatography can be carried out using a gradient which began with 100% water and ended with 100% organic solvent; the chromatography was preferably run with a linear gradient of from 5 to 95% acetonitrile.
  • a gel chromatography or chromatography on hydrophobic phases.
  • the gel chromatography can e.g. be carried out on polyacrylamide gels or copolymer gels.
  • the sequence of the abovementioned chromatographic steps can be reversed.
  • Labyrinthopeptins are present as stereoisomers, they can be separated using known methods, for example by means of separation using a chiral column.
  • the derivatization of the OH group to an ester or ether derivative is effected using methods which are known per se (J. March, Advanced Organic Chemistry, John Wiley & Sons, 4th edition, 1992), for example by means of reaction with an acid anhydride or by reaction with an di-alkyl carbonate or di-alkyl sulfate.
  • Derivatization of the COOH group to an ester or amid derivative is effected using methods which are known per se (J. March, Advanced Organic Chemistry, John Wiley & Sons, 4th edition, 1992), for example by means of reaction with ammonia to the respective CONH 2 group, or with an optionally activated alkyl compound to the respective alkyl ester.
  • Oxidation of -CH 2 -S-CH 2 - groups to a -CH 2 -S(O)-CH 2 - or a -CH 2 -S(O) 2 -CH 2 - group can be achieved upon exposing the respective Labyrinthopeptin derivative to oxygen or air.
  • Reduction of disulfides, optionally followed by alkylation of free SH groups is effected using methods which are known per se (A. Henschen, Analysis of cyst(e)ine residues, disulfide bridges, and sulfhydryl groups in proteins, in: B. Wittmann-Liebold, J. Salnikov, V.A.
  • a mutant is a microorganism in which one or more genes in the genome has/have been modified, with the gene, or the genes, which is/are responsible for the ability of the organism to produce the compound according to the invention remaining functional and heritable.
  • Such mutants can be produced, in a manner known per se, using physical means, for example irradiation, as with ultraviolet rays or X-rays, or chemical mutagens, such as ethyl methanesulfonate (EMS); 2-hydroxy-4-methoxybenzophenone (MOB) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), or as described by Brock et al. in "Biology of Microorganisms", Prentice Hall, pages 238-247 (1984).
  • EMS ethyl methanesulfonate
  • MOB 2-hydroxy-4-methoxybenzophenone
  • MNNG N-methyl-N'-nitro-N-nitrosoguanidine
  • a variant is a phenotype of the microorganism.
  • Microorganisms have the ability to adapt to their environment and therefore exhibit highly developed physiological flexibility. All the cells of the microorganism are involved in the phenotypic adaptation, with the nature of the change not being genetically conditioned and being reversible under altered conditions (H. Stolp, Microbial ecology: organism, habitats, activities. Cambridge University Press, Cambridge, GB, page 180, 1988).
  • Screening for mutants and/or variants which synthesize one or more of the compounds according to the invention is achieved by optionally lyophilizing the fermentation medium and extracting the lyophilizate or the fermentation broth with an organic solvent or a mixture of water and an organic solvent as defined above, and analyzing by means of HPLC or TLC or by testing the biological activity.
  • the fermentation conditions may be applied to Actinomadura namibiensis (DSM 6313) and for mutants and/or variants thereof.
  • a further embodiment of the present invention is the use of a compound of the formula (I), as defined above, for the treatment of bacterial infections, especially bacterial infections caused by Gram-positive bacteria, for the treatment of viral infections and/or for the treatment of pain, especially neuropathic pain or inflammatory triggered pain.
  • the above described medicament (also referred to as pharmaceutical preparation or pharmaceutical composition) contains an effective amount of at least one compound of the formula (I), in any stereochemical form, or a mixture of any stereochemical forms in any ratio, or a physiologically tolerable salt or chemical equivalent thereof, as described above, and at least one pharmaceutically acceptable carrier, preferably one or more pharmaceutically acceptable carrier substances (or vehicles) and/or additives (or excipients).
  • the medicament can be administered orally, for example in the form of pills, tablets, lacquered tablets, coated tablets, granules, hard and soft gelatine capsules, solutions, syrups, emulsions, suspensions or aerosol mixtures.
  • Administration can also be carried out rectally, for example in the form of suppositories, or parenterally, for example intravenously, intramuscularly or subcutaneously, in the form of injection solutions or infusion solutions, microcapsules, implants or rods, or percutaneously or topically, for example in the form of ointments, solutions or tinctures, or in other ways, for example in the form of aerosols or nasal sprays.
  • the medicaments according to the invention are prepared in a manner known per se and familiar to one skilled in the art, pharmaceutically acceptable inert inorganic and/or organic carrier substances and/or additives being used in addition to the compound(s) of the formula (I) in any stereochemical form, or a mixture of any stereochemical forms in any ratio, or a physiologically tolerable salt or chemical equivalent thereof, as described above.
  • pharmaceutically acceptable inert inorganic and/or organic carrier substances and/or additives being used in addition to the compound(s) of the formula (I) in any stereochemical form, or a mixture of any stereochemical forms in any ratio, or a physiologically tolerable salt or chemical equivalent thereof, as described above.
  • lactose corn starch or derivatives thereof, talc, stearic acid or its salts, etc.
  • Carrier substances for soft gelatine capsules and suppositories are, for example, fats, waxes, semisolid and liquid polyols, natural or hardened oils, etc.
  • Suitable carrier substances for the production of solutions, for example injection solutions, or of emulsions or syrups are, for example, water, saline, alcohols, glycerol, polyols, sucrose, invert sugar, glucose, vegetable oils, etc.
  • Suitable carrier substances for microcapsules, implants or rods are, for example, copolymers of glycolic acid and lactic acid.
  • the pharmaceutical preparations normally contain about 0.5 to about 90 % by weight of a compound of the formula (I) and/or their physiologically acceptable salts and/or their prodrugs.
  • the amount of the active ingredient of the formula (I) in any stereochemical form, or a mixture of any stereochemical forms in any ratio, or a physiologically tolerable salt or chemical equivalent thereof, as described above, in the medicaments normally is from about 0.5 to about 1000 mg, preferably from about 1 to about 500 mg.
  • the pharmaceutical preparations can contain one or more additives such as, for example, fillers, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, preservatives, sweeteners, colorants, flavorings, aromatizers, thickeners, diluents, buffer substances, solvents, solubilizers, agents for achieving a depot effect, salts for altering the osmotic pressure, coating agents or antioxidants.
  • additives such as, for example, fillers, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, preservatives, sweeteners, colorants, flavorings, aromatizers, thickeners, diluents, buffer substances, solvents, solubilizers, agents for achieving a depot effect, salts for altering the osmotic pressure, coating agents or antioxidants.
  • the pharmaceutical preparations can also contain two or more compounds of the formula (I) in any stereochemical form, or a mixture of any stereochemical forms in any ratio, or a physiologically tolerable salt or chemical equivalent thereof.
  • a pharmaceutical preparation contains two or more compounds of the formula (I)
  • the selection of the individual compounds can aim at a specific overall pharmacological profile of the pharmaceutical preparation. For example, a highly potent compound with a shorter duration of action may be combined with a long-acting compound of lower potency.
  • the flexibility permitted with respect to the choice of substituents in the compounds of the formula (I) allows a great deal of control over the biological and physico-chemical properties of the compounds and thus allows the selection of such desired compounds.
  • the pharmaceutical preparations can also contain one or more other therapeutically or prophylactically active ingredients.
  • the dose can vary within wide limits and, as is customary and is known to the physician, is to be suited to the individual conditions in each individual case. It depends, for example, on the specific compound employed, on the nature and severity of the disease to be treated, on the mode and the schedule of administration, or on whether an acute or chronic condition is treated or whether prophylaxis is carried out.
  • An appropriate dosage can be established using clinical approaches well known in the medical art.
  • the daily dose for achieving the desired results in an adult weighing about 75 kg is from about 0.01 to about 100 mg/kg, preferably from about 0.1 to about 50 mg/kg, in particular from about 0.1 to about 10 mg/kg, (in each case in mg per kg of body weight).
  • the daily dose can be divided, in particular in the case of the administration of relatively large amounts, into several, for example 2, 3 or 4, part administrations. As usual, depending on individual behaviour it may be necessary to deviate upwards or downwards from the daily dose indicated.
  • Example 1 Preparation of a cryoculture of Actinomadura namibiensis (DSM 6313)
  • 100 ml culture medium (10 g starch, 2 g yeast extract, 10 g glucose, 10 g glycerine, 2.5 g cornsteep powder, 2 g peptone, 1 g NaCI, 3g CaCO 3 in 1 I tap water, pH 7.2 before sterilization) were seeded with the strain Actinomadura namibiensis (DSM 6313) in a sterile 500 ml Erlenmeyer flask and incubated for 72 hours at 27°C and 120 rpm on a shaker.
  • DSM 6313 strain Actinomadura namibiensis
  • 1 ml of the culture and 1 ml sterile conservation solution (20 g glycerine, 10 g saccharose, 70 ml de-ionised water) were mixed and stored at -80 0 C.
  • small pieces of a well-grown culture on agar were transferred into Cryotubes® (Vangard International) with 1.5 ml 50% sterile glycerine solution and stored at -196°C in liquid nitrogen.
  • a sterile 500 ml Erlenmeyer flask containing 100 ml of the culture medium described in Example 1 was seeded with a culture of Actinomadura namibiensis (DSM 6313) which was grown on an agar plate and was incubated at 27°C and 120 rpm on a shaker. After 72 hours, further Erlenmeyer flasks containing the same culture medium in the same amount were seeded with 2 ml of this pre-culture each and incubated under identical conditions for 168 hours.
  • DSM 6313 Actinomadura namibiensis
  • a 300 ml Erlenmeyer flask containing 100 ml of the culture medium described in Example 1 was seeded with a culture of Actinomadura namibiensis (DSM 6313) and incubated at 25 0 C and 180 rpm. After 72 hours, further Erlenmeyer flasks containing the same culture medium in the same amount were seeded with 5 ml of this pre-culture each and incubated under identical conditions for 168 hours.
  • the culture broth After completion of a 40 L-fermentation of Actinomadura namibiensis (DSM 6313) the culture broth has been filtered.
  • the culture filtrate (ca. 30 L) has been loaded onto a column (dimension: 160 x 200 mm) filled ca. 3 L of CHP-20P material.
  • Compounds were eluted at a flow rate of 250 ml/min using a gradient from 5% to 95% of isopropanol in water. Fractions have been collected every 4 min over a period of 45 min.
  • Example 5 Final purification of Labyrinthopeptin A1 Fractions 21-22 from Example 4 (60 mg) have been dissolved in 50 ml methanol and loaded onto a Phenomenex Luna® 5 ⁇ C18 (2) Axia column (dimension: 30 mm x 100 mm) with a Waters XTerra® Prep MS C18 10 ⁇ pre-column (dimension: 19 x 10 mm). Compounds were eluted with a gradient from 5% to 75% acetonithle in water over a period of 40 min at a flow rate of 70 ml/min (buffer: 0.1 % ammonium acetate, pH 4.6, adjusted using aqueous acetic acid).
  • Labyrinthopeptin-containing fractions (f. 9-12) have been pooled. After freeze-drying, 17 mg of Labyrinthopeptin A1 have been obtained.
  • Fraction 8 ( ⁇ 850 mg) from example 3 has been dissolved in 500 ml methanol and loaded onto a Phenomenex Luna® 10 ⁇ C18 (2) column (dimension: 50 mm x 250 mm) with a Phenomenex Luna® 10 ⁇ C18 (2) pre-column (dimension: 21.2 mm x 60 mm).
  • Compounds were eluted with a gradient from 5% to 75% acetonitrile in water over a period of 40 min (buffer: 0.1 % ammonium acetate, pH 7.0) at a flow rate of 190 ml/min. Fractions were collected every minute.
  • Fraction 19 from example 6 (48 mg) has been dissolved in 50 ml methanol and loaded onto a Phenomenex Luna® 5 ⁇ C18 (2) Axia column (dimension: 30 mm x 100 mm) with a Waters XTerra® Prep MS C18 10 ⁇ pre-column (dimension: 19 mm x 10 mm).
  • Compounds were eluted with a gradient from 5% to 75% acetonitrile in water over a period of 40 min at a flow rate of 70 ml/min (buffer: 0.1 % ammonium acetate, pH 9.0, adjusted using a 30% aqueous ammonia solution). The eluents have been collected in fractions using UV-triggering.
  • Labyrinthopeptin-containing fractions (F9-12) have been pooled. After freeze-drying, 12 mg of Labyrinthopeptin A3 have been obtained.
  • Example 8 Characterisation of Labyrinthopeptins A1 and A3 by high performance liquid chromatography with diode-array and mass spectrometry detection (HPLC-DAD- MS)
  • Labyrinthopeptins A1 and A3 were analyzed on a Waters Acquity UPLC System with Sample Manager, Binary Solvent Manager and PDA (Photodiode Array Detector).
  • UPLC column a Waters Acquity UPLC BEH C18 (1.7 ⁇ ; 2.1x100 mm) was used and eluted at a flow rate of 0.6 ml/min with a gradient of wate ⁇ acetonitrile (9:1) within 15 min to 100 % acetonitrile, all solvents buffered with 6.5mM ammonium acetate to pH 4.6.
  • UV spectra were recorded by the PDA detector at wavelengths between 200 and 600 nm.
  • Mass spectra were recorded with a Bruker ⁇ TOF LC MS using an orthogonal electrospray ionisation, a sampling-rate of 0.5 Hz and a detection-limit of 150-1500 atomic mass units.
  • Labyrinthopeptin A1 eluted at 5.46 min (PDA).
  • the UV spectrum is featured by ⁇ max of 218 nm (sh) and 279 nm.
  • Labyrinthopeptin A3 eluted at 4.79 min (PDA).
  • the UV spectrum is featured by ⁇ max of 218 nm (sh) and 274 nm (sh).
  • Labyrinthopeptin A1 (0.05 mg) was hydrolyzed in nitrogen atmosphere with 6 N HCI, 5% phenole at 110 0 C for 24 h. The hydrolysate was dried in a stream of nitrogen.
  • Achiral GC-MS The hydrolysate was heated with bis-(trimethylsilyl)trifluoroacetamide (BSTFA)/acetonitrile (1 :1) at 150 0 C for 4 h.
  • BSTFA bis-(trimethylsilyl)trifluoroacetamide
  • Example 12 Identification of the structural genes for Labyrinthopeptins A1 and A3
  • a cosmid bank of the microorganism Actinomadura namibiensis (DSM 6313) was generated by Agowa GmbH, Berlin, based on the pWEB-cosmid vector (Epicentre Biotechnologies, Madison, USA). Filters were prepared by RZPD GmbH, Berlin, applying a methodology described in: Zehetner & Schafer, Methods MoI. Biol. 2001 , 175, 169-188.
  • the 5 ' -elongation of the primers was to enhance the expected PCR-product size for better detection and handling (PCR-conditions: 3 min 95 °C; 30 x (60 s 95 0 C; 30 s 50 °C; 60 s 72 0 C) 7 min 72 °C; Taq-polymerase).
  • the PCR-product was gel-purified and cloned into the vector pDrive (Qiagen). Sequencing resulted in a 18 nucleotide length sequence from the middle of the A2 gene (AGTGCTGTAGCACGGGAA, SEQ ID NO: 6). Based on this 18 nucleotide long known sequence, a two-step PCR rendered to more sequence information. In the first step, a single-specific primer-PCR was performed with a degenerated reversed(rev)-primer of the C-terminal end of A2
  • the PCR-sample was used as template for a second PCR (PCR-conditions: 3 min 95°C; 30 x (45 s 95°C; 45 s 56°C; 3,5 min 72°C) 5 min 72°C; Taq-polymerase).
  • the second PCR was performed in a nested- PCR manner with a primer pair consisting of the unspecific fw-primer from the first PCR and a specific rev-primer, including the known 18 nucleotides
  • the propeptide sequence is transformed into Labyrinthopeptin A2 by posttranslational modifications by enzymes of the microorganism Actinomadura namibiensis (DSM 6313).
  • Example 13 Structure determination of Labyrinthopeptins A1 and A3:
  • the upstream region of the A2 gene displays another small orf with high homology to the structural gene of Labyrinthopeptin A2.
  • This open reading frame (orf) included the structural gene of Labyrinthopeptin A1 and A3.
  • the orf for Labyrinthopeptin A1 has the following gene sequence:
  • Example 14 Cleavage of the disulfide-bridge of Labyrinthopeptin A1 and subsequent alkylation with methyliodide
  • Labyrinthopeptin A1 (50 mg, 0.024 mmol) was dissolved in methanol (3 ml) and a dithiothreitol solution was added at room temperature (1 ml, freshly prepared from 75 mg dithiothreitol in a solution of 40 mg NaHCO 3 in 1 ml water). The mixture was stirred for 1 h at 60 0 C. Afterwards it was cooled down to room temperature and methyliodide (50 ⁇ l, 0.80 mmol) was added.
  • Example 15 Cleavage of the disulfide-bridge of Labyrinthopeptin A1 and subsequent alkylation with iodo-acetamide
  • Labyrinthopeptin A1 (50 mg, 0.024 mmol) was dissolved in methanol (3 ml) and a dithiothreitol solution was added at room temperature (1 ml, freshly prepared from 70 mg dithiothreitol in a solution of 40 mg NaHCOs in 1 ml water). The mixture was stirred for 1 h at 6O 0 C. Afterwards it was cooled down to room temperature and iodo- acetamide (40 mg, 0.216 mmol) was added. The mixture was stirred over night at room temperature.
  • the solution was filtered and purified by reversed-phase HPLC using a Phenomenex Luna® Axia 5 ⁇ m C18 (2) column (dimension: 100 mm x 30 mm) with a Waters XTerra® Prep MS C18 10 ⁇ m pre-column (dimension: 19 mm x 10 mm).
  • the gradient was running from 5% to 95% acetonithle in water within 30 minutes (buffer: 0.1% ammonium acetate, pH 4.6, adjusted with acetic acid).
  • the flow was 60 ml/min and the peaks were fractionated by UV.
  • the following compounds were obtained:
  • the mixture was purified by reversed-phase HPLC using a Waters XBridge Shield® 5 ⁇ m C18 column (dimension: 100 mm x 30 mm) with a Waters XBridge Shield® C18 10 ⁇ m pre-column (dimension: 19 mm x 10 mm).
  • the gradient was running from 5% to 95% acetonitrile in water within 30 minutes (buffer: 0.1% ammonium acetate, pH 7.0).
  • the flow was 60 ml/min and the peaks were fractionated by UV (220 nm). Fraction 39 yielded 2.0 mg (3.8%) of the desired compound after lyophilization.
  • the product was characterized by UV spectroscopy and mass spectrometry (Bruker Daltonics MicroTof).
  • the compounds were dissolved in water with 10 % MeOH to a final concentration of 1 mg/ml.
  • sterile Nunc plates with a size of 24 x 24 cm were used.
  • 200 ml of agar were used.
  • the agar was cooled after autoclaving to 55°C and 2-4 ml of culture suspension of the test organism were added before plating.
  • Labyrinthopeptin A1 was studied in the spared nerve injury (SNI) mouse model of neuropathic pain in order to proof the activity on tactile allodynia.
  • SNI spared nerve injury
  • the two major branches of the sciatic nerve in adult male C57B6 mice have been ligated and transsected, with the sural nerve left intact.
  • Tactile allodynia has been determined with the automatic von Frey test: using a dump needle stick, the plantar skin of hind paws was exposed to a pressure stimulus of increasing intensity up to 5 g. The force in grams at which the animal responded with hindpaw withdrawal was used as a read-out for tactile allodynia.
  • the compound was administered intravenous as a single application (3 mg/kg).
  • As a vehicle for the intravenous application was the 1 :1 :18 (ethanoksolutol: phosphate buffered saline) vehicle chosen.
  • Paw withdrawal threshold (PWT) measurements have been used to calculate significant treatment effects, and for AUC calculations over a reference time period (6 hours) and subsequent % benefit calculations.
  • PWT values of the ipsilateral hind paws were used in two ways: first, with a 2-way ANOVA based on the PWT values for specific times (within a period of 24 hours) and second with a 1-way ANOVA on non-transformed delta AUC values
  • the percent benefit of the treatment was evaluated using the
  • values of the contralateral sides of all three groups (100% benefit maximal possible effect). Compared to these margins Labyrinthopeptin A1 achieved 95% benefit.
  • the compounds of the formula (I) significantly reduce tactile allodynia in the SNI mouse model of neuropathic pain.

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EP09728150A 2008-04-02 2009-03-18 Stark verbrückte peptide aus der actinomadura namibiensis Not-in-force EP2271665B1 (de)

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EP1908774A1 (de) * 2006-10-06 2008-04-09 sanofi-aventis Antibakterielle und antivirale Peptide aus Actinomadura namibiensis
DK2257301T3 (da) 2008-03-03 2014-04-28 Univ Miami Immunterapi baseret på allogene cancerceller.
KR20110017356A (ko) 2008-03-20 2011-02-21 유니버시티 오브 마이애미 열충격 단백질 ｇｐ96 백신접종 및 이를 사용하는 방법
EP2607374A1 (de) * 2011-12-21 2013-06-26 Sanofi Verfahren zur rekombinanten Herstellung von Labyrinthopeptinen und funktionalen Derivaten davon
US10487124B2 (en) * 2012-07-12 2019-11-26 Naicons S.R.L. Lantipeptide
US20190046604A1 (en) 2015-11-18 2019-02-14 Helmholtz-Zentrum für Infektionsforschung GmbH Labyrinthopeptins as anti-viral agents

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EP0700998B1 (de) * 1994-09-12 2003-11-26 Aventis Pharma Deutschland GmbH Rekombinantes Mersacidin und Verfahren zu seiner Herstellung
US5942423A (en) 1995-06-07 1999-08-24 Massachusetts Institute Of Technology Conversion of compactin to pravastatin by actinomadura
JP4664924B2 (ja) * 2003-12-02 2011-04-06 ウーツェーベー ファルマ ゲーエムベーハー 中枢神経因性疼痛の治療のためのペプチド化合物の新規使用
CN100516218C (zh) * 2006-04-30 2009-07-22 中山大学 一种新的线纹芋螺毒素序列、制备方法及其应用
EP1908774A1 (de) * 2006-10-06 2008-04-09 sanofi-aventis Antibakterielle und antivirale Peptide aus Actinomadura namibiensis
EP2607374A1 (de) * 2011-12-21 2013-06-26 Sanofi Verfahren zur rekombinanten Herstellung von Labyrinthopeptinen und funktionalen Derivaten davon

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IL208239A0 (en) 2010-12-30
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KR20110014563A (ko) 2011-02-11
ATE541856T1 (de) 2012-02-15
CA2720173A1 (en) 2009-10-08
KR101612275B1 (ko) 2016-04-14
WO2009121483A1 (en) 2009-10-08
CN102046652A (zh) 2011-05-04
AU2009231118B2 (en) 2013-06-27
JP5653900B2 (ja) 2015-01-14
CL2009000791A1 (es) 2009-10-09
CN102046652B (zh) 2014-11-05
AU2009231118A1 (en) 2009-10-08
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MY159790A (en) 2017-01-31
EP2271665B1 (de) 2012-01-18
HK1155762A1 (en) 2012-05-25

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